1. Field of the Invention
The invention relates to oligonucleotide-quencher-fluorescent-dye conjugates having improved characteristics, and to reagents suitable for incorporating novel quencher and fluorescent dye moieties into oligonucleotides. The invention also relates to the use of oligonucleotide-quencher-fluorescent-dye conjugates in detection methods for nucleic acid targets.
2. Brief Description of Related Art
Synthetic oligonucleotides have been used for years as sequence specific probes for complementary DNA and RNA targets. These methods have broad application in forensics, molecular biology and medical diagnostics since they allow the identification and quantitation of specific nucleic acid targets. Early uses of DNA probes relied on radioactivity (typically 32P) as the label, while recent methods use reporter molecules which include chemiluminescent and fluorescent groups. Improved instrumentation has allowed the sensitivity of these spectroscopic methods to approach or surpass the radiolabeled methods. Recently developed detection methods employ the process of fluorescence resonance energy transfer (FRET) for the detection of probe hybridization rather than direct detection of fluorescence intensity. In this type of assay, FRET occurs between a donor fluorophore (reporter) and an acceptor molecule (quencher) when the absorption spectrum of the quencher molecule overlaps with the emission spectrum of the donor fluorophore and the two molecules are in close proximity. The excited-state energy of the donor fluorophore is transferred to the neighboring acceptor by a resonance dipole-induced dipole interaction, which results in quenching of the donor fluorescence. If the acceptor molecule is a fluorophore, its fluorescence may sometimes be increased. The efficiency of the energy transfer between the donor and acceptor molecules is highly dependent on distance between the molecules. Equations describing this relationship are known. The Forster distance (Ro) is described as the distance between the donor and acceptor molecules where the energy transfer is 50% efficient. Other mechanisms of fluorescence quenching are also known, such as, collisional and charge transfer quenching.
Typically detection methods based on FRET are designed in such a way that the donor fluorophore and acceptor molecules are in close proximity so that quenching of the donor fluorescence is efficient. During the assay, the donor and acceptor molecules are separated such that fluorescence occurs. FRET-based detection assays have been developed in the fields of nucleic acid hybridization and enzymology. Several forms of the FRET hybridization assays are reviewed (Nonisotopic DNA Probe Techniques, Academic Press, Inc., San Diego 1992, pp. 311-352). Quenching can also occur through non-FRET mechanisms, such as collisional quenching (see, Wei, et al., Anal. Chem. 66:1500-1506 (1994)).
Since its discovery, the polymerase chain reaction (PCR) has revolutionized molecular biology. This technique allows amplification of specific DNA sequences, thus allowing DNA probe assays to be executed from a single DNA target copy. PCR-based diagnostic assays have initially not been used routinely, in part due to problems with sample handling and possible contamination with non-source DNA. Recently, new homogeneous fluorescent-based DNA assays have been described which can detect the progress of PCR as it occurs (xe2x80x9creal-timexe2x80x9d PCR detection) using spectrofluorometric temperature cyclers. Two popular assay formats use DNA probes which become fluorescent as DNA amplification occurs (fluorogenic probes).
The first format for xe2x80x9creal-timexe2x80x9d PCR uses DNA probes known as xe2x80x9cmolecular beaconsxe2x80x9d (Tyagi et al., Nat. Biotech., 16: 49-53 (1998)). Molecular beacons have a hairpin structure wherein the quencher dye and reporter dye are in intimate contact with each other at the end of the stem of the hairpin. Upon hybridization with a complementary sequence, the loop of the hairpin structure becomes double stranded and forces the quencher and reporter dye apart, thus generating a fluorescent signal. Tyagi et al. reported use of the non-fluorescent quencher dyes including the dabcyl (4-{[4-(dimethylamino)phenyl]diazenyl}benzoyl moiety, absorbance max=453 nm) used in combination with fluorescent reporter dyes of widely varying emission wavelength (475-615 nm). At the time this was surprising since FRET requires significant overlap of the absorption spectrum of the quencher and of the emission spectrum of the reporter. In case of a dabcyl moiety containing (hereinafter xe2x80x9cdabcylxe2x80x9d) quencher and some fluorescent dyes, the spectral overlap was extremely low, yet quenching efficiency was high. Therefore it was proposed that the mechanism of quenching for the hairpin form of the beacons was not FRET, but collisional quenching. In fact, the UV spectra of the quencher changes in the hairpin form of the beacon, providing evidence of the molecular contact and thus of collisional quenching. A related detection method uses hairpin primers as the fluorogenic probe (Nazarenko et al., Nucl. Acid Res., 25: 2516-2521 (1997)).
The second format for xe2x80x9creal-timexe2x80x9d PCR uses DNA probes which are referred to as xe2x80x9c5xe2x80x2-nuclease probesxe2x80x9d (Lee et al., Nucl. Acid Res., 21: 3761-3766 (1993)). These fluorogenic probes are typically prepared with the quencher at the 3xe2x80x2 terminus of a single DNA strand and the fluorophore at the 5xe2x80x2 terminus. During each PCR cycle, the 5xe2x80x2-nuclease activity of Taq DNA polymerase cleaves the DNA strand, thereby separating the fluorophore from the quencher and releasing the fluorescent signal. The 5xe2x80x2-nuclease assay requires that the probe be hybridized to the template strand during the primer extension step (60-65xc2x0 C.). They also disclose the simultaneous xe2x80x9creal-timexe2x80x9d detection of more than one polynucleotide sequence in the same assay, using more than one fluorophore/quencher pair. The 5xe2x80x2-nuclease PCR assay is depicted in FIG. 1.
Initially it was believed that 5xe2x80x2-nuclease probes had to be prepared with the quencher (usually tetramethylrhodamine (TAMRA)) positioned at an internal nucleotide in close proximity to the 5xe2x80x2-fluorophore (usually fluorescein (FAM) or tetrachlorofluorescein (TET)) to get efficient FRET. Later it was found that this is not necessary, and the quencher and the fluorophore can be located at the 3xe2x80x2 and 5xe2x80x2 end of the ODN, respectively. It has been proposed that the random coil structures formed by these fluorogenic probes in solution allow a 3xe2x80x2-quencher dye to pass within the Forster radius of the 5xe2x80x2-fluorophore during the excited state of the molecule.
A number of donor/acceptor pairs have previously been described, important to the present invention is dabcyl that is used for instance as a quencher of dansyl sulphonamide in chemosensors (Rothman and Still (1999) Med. Chem. Lett. 22:509-512).
Surprisingly, there have been no published reports on the use of dabcyl in 5xe2x80x2-nuclease probes or other FRET probes that use long wavelength fluorophores. As mentioned above, dabcyl was used in the beacon-type probes but this is a different quenching mechanism wherein the dabcyl and fluorophore are in intimate contact (collisional quenching). Dabcyl was used in fluorogenic peptides as a quencher for the fluorophore EDANS (5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid) which emits at short (490 nm , blue) wavelength (Matayoshi et al. Science 247: 954-958 (1990)). EDANS also has a lower extinction coefficient than dabcyl so it is not surprising that fluorescent quenching was efficient. It was found for the first time in the present invention that dabcyl can be used to quench fluorescein in a FRET type mechanism.
In addition to the 5xe2x80x2-nuclease PCR assay, other formats have been developed that use the FRET mechanism. For example, single-stranded signal primers have been modified by linkage to two dyes to form a donor/acceptor dye pair in such a way that fluorescence of the first dye is quenched by the second dye. This signal primer contains a restriction site (U.S. Pat. No. 5,846,726) that allows the appropriate restriction enzyme to nick the primer when hybridized to a target. This cleavage separates the two dyes and a change in fluorescence is observed due to a decrease in quenching. Non-nucleotide linking reagents to couple oligonucleotides to ligands have also been described (U.S. Pat. No. 5,696,251).
FRET systems also have applications in enzymology. Protease cleavable substrates have been developed where donor/acceptor dye pairs are designed into the substrate. Enzymatic cleavage of the substrate separates the donor/acceptor pair and a change in fluorescence is observed due to a decrease in quenching. Cleavable donor/acceptor substrates have been developed for chymotrypsin (Li et al. Bioconj. Chem., 10: 241-245 (1999)), aminopeptidase P (Hawthorne et al., Anal. Biochem., 253: 13-17 (1997)), stromelysin (Bickett et al., Ann. N. Y. Acad. Sci., 732: 351-355 (1994)) and leukotriene D4 hydrolase (White et al., Anal. Biochem., 268: 245-251 (1999)). A chemosensor was described where binding of the ligand separates the donor/acceptor pair (Rothman et al. Biorg. Med. Chem. Lett., 9: 509-512 (1999)).
U.S. Pat. No. 5,801,155 discloses that oligonucleotides (ODNs) having a covalently attached minor groove binder (MGB) are more sequence specific for their complementary targets than unmodified oligonucleotides. In addition the MGB-ODNs show substantial increase in hybrid stability with complementary DNA target strands when compared to unmodified oligonucleotides, allowing hybridization with shorter oligonucleotides.
Reagents for fluorescent labeling of oligonucleotides are critical for efficient application of the FRET assays described above. Other applications such as DNA micro arrays also use fluorescently labeled DNA probes or primers, and there is a need for improved reagents which facilitate synthesis of fluorescent DNA. In general, phosphoramidite reagents and solid supports are widely used on ODN synthesis. However, there are few commercially available phosphoramidite reagents for introducing fluorescent groups into ODNs.
Linker groups to attach different ligand groups to ODNs play an important role in the synthesis of oligonucleotide conjugates. A method for the synthesis of 3xe2x80x2-aminohexyl-tailed oligonucleotides (Petrie et al., Bioconj. Chem., 3:85-87 (1992)), the use of a trifunctional trans-4-hydroxy-L-prolinol group (Reed et al., Bioconjug. Chem., 2:217-225 (1991)), diglycolic acid (Pon et al., Nucl. Acids. Res., 25:3629-3635 (1997)), 1,3-diol reagents (U.S. Pat. Nos. 5,942,610 and 5,451,463) and a non-nucleotide trifunctional reagent (U.S. Pat. No. 5,696,251) have been reported.
Resorufin and coumarin derivatives have been extensively used as enzyme substrates to differentiate isozymes of cytochrome P450 (Haugland et al., Handbook of Fluorescent Probes and Research Chemicals, Six Edition, Eugene, Org. pp. 235-236. 1996.). Reactive resorufin analogs have been disclosed in U.S. Pat. No. 5,304,645. Activated esters of coumarin derivatives are also known in the art (Hirshberg et al., Biochem., 37:10391-5 (1998)). Coumarin-labeled dUTP incorporated in probes were used for in situ hybridizations (Wiegant et al., Cytogenet. Cell Genet., 63:73-76 (1993)). Phosphoramidites to introduce labels into oligonucleotides have been described in U.S. Pat. Nos. 5,328,824 and 5,824,796.
Many current hybridization applications, require more than one reporter molecule. In addition although reporter fluorophores are available to be used in reporter/quencher pairs, most suffer from having some undesirable characteristics, e.g. mixtures are difficult to separate, they are positively charged or difficult to synthesize, unstable during oligonucleotide synthesis or having overlapping emission wavelengths with other desirable reporters.
The present invention provides reagents for oligonucleotide probes that address these unfavorable characteristics and overcome some or all of the difficulties.
In one aspect, the present invention provides an oligonucleotide probe having the formula: 
wherein Ar1 and Ar2 each independently represent a susbstituted or unsubstituted aryl group; MGB is a minor groove binding group; FL is a fluorescent group having an emission maxima in the region from about 400 to about 900 nm; K is a cyclic or acyclic linking group having from 1 to 30 backbone atoms selected from C, N, O, S and P; W is a linking group having from 3 to 100 backbone atoms selected from C, N, O, S, Si and P which is cyclic, acyclic, aromatic or a combination thereof; [Axe2x80x94B]n is a natural or modified oligonucleotide having from 4 to 100 bases; and the subscript n is an integer of from 4 to 100.
In one group of embodiments, Ar1 is a substituted or unsubstituted aryl group selected from the group consisting of phenyl, naphthyl, 2-benzothiazolyl, 3-benzoisothiazolyl and 2-thiazolyl. In another group of embodiments, Ar2 bears from one to three substituents selected from nitro, cyano, halo, xe2x80x94C(O)R1, xe2x80x94C(O)NR1R2, xe2x80x94SO2R1, xe2x80x94SO2F and xe2x80x94SO2NR1R2, wherein each R1 and R2 is independently H, (C1-C6)alkyl or hydroxy(C1-C6)alkyl. In still other embodiments, the group xe2x80x94Ar1xe2x80x94Nxe2x95x90Nxe2x80x94Ar2 is a quencher moiety having the formula: 
wherein R0, R1, R2, R3 and R4 are independently selected from H, halogen, NO2, SO3R, SO2N(R)2, C(O)OR, C(O)N(R)2, CN, CNS, OR, OC(O)R, SR, CF3, NHC(O)R, N(R)2 and N[R]3 wherein each R is independently selected from H, (C1-C8)alkyl, aryl (and heteroaryl), or a blocking group compatible with oligonucleotide synthesis; and R5 is xe2x80x94H or (C1-C8)alkyl, and the quencher moiety is attached to the linker through the valence bond designated xe2x80x9caxe2x80x9d. In still other embodiments, the group Wxe2x80x94Ar1xe2x80x94Nxe2x95x90Nxe2x80x94Ar2 is a quencher moiety-linking group combination having a formula selected from Q-1, Q-2 and Q-3: 
wherein q, r, s, t and v are each independently an integer of from 1 to 20; X is xe2x80x94Oxe2x80x94, xe2x80x94OCH2xe2x80x94 or xe2x80x94CH2xe2x80x94; and the conjugated quencher and linker moiety is attached to the [Axe2x80x94B]n portion through one of the valence bonds designated a or b; and is attached to the minor groove binder portion through the other of valence bonds designated a or b.
In still other embodiments, the oligonucleotide probe has a fluorophore, FL selected from the group of FL-1, Fl-2 and Fl-3: 
wherein R21, R22, R23, R24, R25, R26 and R27 are each substituents independently selected from H, halogen, NO2, SO3R, SO2N(R)2, C(O)OR, C(O)N(R)2, CN, CNS, OR, OC(O)R, SR, CF3, NHC(O)R, N(R)2 and N[R]3 wherein each R is independently selected from the group consisting of H, (C1-C8)alkyl, aryl (and heteroaryl), or a blocking group compatible with oligonucleotide synthesis, and optionally two adjacent groups from R21 through R26 are combined to form a five- or six-membered ring having from zero to three heteroatoms as ring member, with the proviso that at least one of R21 through R27 is a bond that attaches said fluorophore to said linking group K; and R28 is a member selected from the group consisting of H and (C1-C8)alkyl.
In still other embodiments, the oligonucleotide probes of the invention comprise a minor groove binder (MGB) that is selected from analogs of one of the following: CC1065, Hoeschst 33258, DAPI, lexitropsins, distamycin, netropsin, berenil (and related diarylamidines), duocarmycin, pentamidine, 4,6-diamino-2-phenylindole, and pyrrolo[2,1-c][1,4]benzodiazepines.
In preferred embodiments, the probes have attached novel quencher structures (described below), paired with a covalently attached fluorescent moiety. The resulting FL-ODN-Q conjugate will preferably include a minor groove binder (MGB) that improves the binding and discrimination characteristics of the resulting FL-ODN-Q-MGB conjugate. These conjugates find particular utility in diagnostic assays such as the TaqMan(copyright) PCR assay for single nucleotide polymorphisms (and the like) where allele-specific discrimination not only requires probes with different fluorescent reporter molecules but efficient quenchers. The quenchers used in the FL-ODN-Q-MGB conjugates are preferably those that provide a broad quenching wavelength range. Additionally, the novel reporter labeling reagents used to prepare these conjugates are those that have distinctive emission wavelengths for improved multicolor analysis.
In one application of the principles summarized above, fluorogenic probes are prepared using a universal xe2x80x9c3xe2x80x2-hexanolxe2x80x9d solid support (available in accordance with Gamper et al. Nucleic Acids Res., 21:145-150 (1993), where a quencher phosphoramidite of the invention is added at the first coupling step (3xe2x80x2-end) of the ODN sequence and a fluorophore (FL) is attached at the final coupling step, yielding 5xe2x80x2-FL-ODN-Q-hexanol conjugate probes.
In another aspect, the present invention provides a quencher reagents having the formula: 
wherein W is a linking group having from 3 to 100 main chain atoms selected from C, N, O, S, P and Si and can be acyclic, cyclic or aromatic or combinations thereof; X1 is H, (C1-C12)alkyl, aryl, heteroaryl, protected or unprotected functional group (e.g., a hydroxy, amino or carboxylic acid or ester that optionally is protected with a suitable protecting group as are known to those of skill in the art); X2 is any phosphorus coupling moiety used in oligonucleotide synthesis, for example, a phosphoramidite of the formula Oxe2x80x94P(N(iPr)2)(OCH2CH2CN), or alternatively, a linking group attached to a solid support of the formula Oxe2x80x94C(xe2x95x90O)Z-solid support wherein Z is 1 to 30 main chain atoms in length wherein the main chain atoms are selected from C, N, O, P, and S, and Z can include acyclic, cyclic or aromatic groups or combinations thereof; and R0, R1, R2, R3 and R4 are independently selected from the group consisting of H, halogen, NO2, SO3R, SO2N(R)2, C(O)OR, C(O)N(R)2, CN, CNS, OR, OC(O)R, SR, CF3, NHC(O)R, N(R)2 or N[R]3 wherein each R is independently H, (C1-C8)alkyl, aryl (and heteroaryl), or a cleavable linking group that is attached to a solid support, or a blocking group compatible with oligonucleotide synthesis and optionally, two of R0, R1 and R2 are combined to form a five- or six-membered ring having from zero to three heteroatoms as ring members; and optionally R3 and R4 are combined to form a five- or six-membered ring having from zero to three heteroatoms as ring members. For those embodiments in which R3 and R4 are combined to form a fused ring system, the linking group W can be attached to either the phenyl ring (as indicated above) or to the ring formed by R3 and R4. Additionally, for those embodiments herein, where two alkyl groups are attached to a nitrogen atom, forming a dialkylamino substituent, the alkyl groups can be the same or different. Preferably, the quencher-phosphoramidite reagent has a formula selected from formulas designated PA-1, PA-2 and PA-3
wherein R0, R1, R2, R3 and R4 are each independently selected from H, halogen, NO2, SO3R, SO2N(R)2, C(O)OR, C(O)N(R)2, CN, CNS, OR, OC(O)R, SR, CF3, NHC(O)R, N(R)2 or N[R]3 wherein each R is independently H, (C1-C8)alkyl, aryl (and heteroaryl), or a blocking group compatible with oligonucleotide synthesis; and optionally, two of R0, R1 and R2 are combined to form a five- or six-membered ring having from zero to three heteroatoms as ring members; and optionally R3 and R4 are combined to form a five- or six-membered ring having from zero to three heteroatoms as ring members; R5 is H or (C1-C8)alkyl; the subscripts q, r, s, t and v are each independently an integer of from 1 to 20; X is xe2x80x94Oxe2x80x94 or xe2x80x94CH2xe2x80x94; and X1 is selected from the group consisting of OH, O-dimethoxytrityl, O-methoxytrityl, O-trityl or an oxygen atom having an acid labile blocking group.
The novel quencher reagents are based on the 4-[4-nitrophenyl)diazinyl]phenylamine and/or the 4-[4-nitrophenyl)diazinyl]-naphthylamine structure. In general, other xe2x80x9cdiazoxe2x80x9d quenchers are also useful, such as those quenchers having a Ar1xe2x80x94Nxe2x95x90Nxe2x80x94Ar2 structure wherein Ar1 and Ar2 are each independently substituted or unsubstituted aryl groups such as phenyl, naphthyl, thienyl, benzo[c]isothiazolyl, and the like. One of skill in the are will appreciate that at least one of Ar1 and Ar2 will have a functional group (e.g., hydroxy, amino, thiol, carboxylic acid, carboxamide and the like) that can to used to attach the quencher to an oligonucleotide or to a linking group. Additionally, these quencher molecules have improved UV spectral overlap not only with commonly used fluorescent reporter groups that emit short wavelength range (about 400 to 500 nm), but have extended the range to the mid (525 nm=green) to long (670 nm=red) and longer wavelengths. The quencher chromophores of the present invention are non-fluorescent, easily incorporated into DNA synthesis reagents, stable during automated DNA synthesis and during storage and have compatible properties in hybridization assays. Moreover, improved signal to noise ratios are observed with the fluorescent reporter dyes over a more extended wavelength range. Accordingly, the present invention offers considerable advantages over the use of dabcyl (Nazerenko et al, Nucl. Acids Res., 25:2516-21 (1997)) as a quenching dye.
In another aspect, the xe2x80x9cdiazoxe2x80x9d quenchers above (based on the 4-[4-nitrophenyl)diazinyl]phenylamine and/or the 4-[4-nitrophenyl)diazinyl]-naphthylamine structure, or related structures) are modified with linking groups (or xe2x80x9clinkersxe2x80x9d) that allow not only their easy incorporation into fluorogenic DNA probes during automated DNA synthesis, but also to modulate the wavelength and ability to quench. In a related aspect, phosphoramidite derivatives of these quenchers are provided and are useful for introducing the quencher moieties into oligonucleotides during automated synthesis, or for attaching the quencher moieties to amino-tailed oligonucleotides.
In another related aspect, the novel quencher molecules are introduced into oligonucleotides using pyrazolo[5,4-d]pyrimidine and pyrimidines phosphoramidites containing the quenchers attached at the 3xe2x80x2- and 5xe2x80x2-positions, respectively.
In yet another aspect, the present invention provides a fluorophore-phosphoramidite reagent having the formula: 
wherein K is a bifunctional linking group; and FL is a fluorophore selected from: 
wherein R21, R22, R23, R24, R25, R26 and R27 are each substituents independently selected from H, halogen, NO2, SO3R, SO2N(R)2, C(O)OR, C(O)N(R)2, CN, CNS, OR, OC(O)R, SR, CF3, NHC(O)R, N(R)2 and N[R]3 wherein each R is independently selected from H, (C1-C8)alkyl, aryl (and heteroaryl), or a blocking group compatible with oligonucleotide synthesis, and optionally two adjacent groups from R21 through R26 are combined to form a five- or six-membered ring having from zero to three heteroatoms as ring member, with the proviso that at least one of R21 through R27 is a bond that attaches said fluorophore to said linking group K; the subscripts q, s and t are integers of from 0 to 5; and R28 is selected from H and (C1-C8)alkyl. These fluorescent reagents are compatible with DNA synthesis and are synthesized or selected and converted into phosphoramidite reagents suitable for incorporation onto ODNs. Specifically, violet fluorescent dyes based on the 10-phenyl-1,3,5,7,9,10-hexahydropyrimidino[5xe2x80x2,4xe2x80x2-5,6]pyridino[2,3-d]pyrimidine-2,4,6,8-tetraone (PPT) structure; red fluorescent dyes based on 7-hydroxyphenoxazin-3-one (resorufin); and blue fluorescent dyes based on the structure of coumarin are incorporated into phosphoramidite reagents, and can be used in preparing compositions provided herein. These fluorescent dyes have excellent properties for multicolor fluorescent analysis in combination with other dyes (e.g. fluorescein). These reagents are useful in a variety of analytical methods that use either direct detection of fluorescence or FRET and related detection formats. In a related aspect of the invention the PPT-, coumarin- and resorufin-based fluorophores (fluorescent dyes) are converted into novel reagents suitable for xe2x80x9cpost-oligonucleotide-synthesisxe2x80x9d covalent attachment at the 5xe2x80x2-end of ODNs. In another aspect, the new fluorescent dyes are incorporated into oligonucleotides using pyrazolo[5,4-d]pyrimidine and pyrimidine phosphoramidites which contain the fluorophores attached at the 3- and 5-positions, respectively.
In still another aspect, the present invention provides a method for hybridizing nucleic acids comprising:
a) incubating a first oligonucleotide with an oligonucleotide probe; and
b) identifying a hybridized nucleic acid;
wherein the oligonucleotide probe is a probe as described above. Preferably, the oligonucleotide probe comprises a fluorophore selected from FL-1, FL-2 and FL-3. In one group of embodiments, the method further comprises the step of altering the spatial relationship between the fluorophore and quencher portions of the oligonucleotide probe. In particular, the altering can be a result of hybridization. In other embodiments, the method further comprises releasing the fluorophore from the oligonucleotide probe subsequent to hybridization.
In still other aspects, methods for synthesizing and attaching the novel quenchers to ODN-fluorophore conjugates, with and without a 3xe2x80x2- or 5xe2x80x2-minor groove binder (MGB) are disclosed. In one group of preferred embodiments, these methods utilize solid supports for automated oligonucleotide synthesis with cleavable linkers. One skilled in the art will also appreciate that MGBs can be attached at internal oligonucleotide positions using linking groups or bases suitably modified to incorporate such compounds.
In yet another aspect, a fluorogenic oligonucleotide probe is prepared from a MGB-modified solid support using techniques similar to those described in Lukhtanov et al. Bioconjugate Chem., 7:564-567 (1996). In this aspect, a quencher-phosphoramidite of the invention is added at the first coupling step to the MGB, and a fluorophore (FL) is attached at the final coupling step to the ODN, to yield 5xe2x80x2-FL-ODN-Q-MGB conjugate probe. Alternatively, a 5xe2x80x2-MGB-Q-ODN-FL can be synthesized using a 5xe2x80x2-phosphoramidite rather than a 3xe2x80x2-phosphoramidite.
Still other aspects are directed to methods and compositions that are useful in micro-arrays in nucleic acid-based diagnostic assays which recently have become important in many fields, such as the medical sciences, forensics, agriculture and water quality control. Other related application of the methods and compositions of the present invention are in procedures using arrays of oligonucleotides, such as the array-based analysis of gene expression (Eisen, Methods of Enzym.; 303:179-205 (1999)). In these procedures, an ordered array of oligonucleotides or DNAs that correspond to all, or a large fraction of the genes in many organism is used as a platform for hybridization. Microarray-based methods are used in assays to measure the relative representation of expressed RNA species. The quantitation of differences in abundance of each RNA species is achieved by directly comparing two samples by labeling them with spectrally distinct fluorescent dyes and mixing the two probes for simultaneous hybridization to one array.
To the extent the application of the compositions and methods of present invention relates to the detection of nucleic acids, it includes but is not limited to methods where FRET is involved, such as 5xe2x80x2-nuclease, universal energy transfer primers or beacon assays. These methods are usually directed to, but are not limited to the detection of PCR-generated nucleic acid sequences. Some of these methods involve simultaneous detection of more than one nucleic acid sequence in the same assay. Similarly, the invention relates to methods where FRET and related quenching mechanisms are involved in the detection of protein concentration or enzyme activity.
Still other applications of the invention relate to the labeling with luminescent PPT-, coumarin- and resorufin-based dyes of nucleic acids, proteins and other materials including, drugs, toxins, cells, microbial materials, particles, glass or polymeric surfaces and the like, at a reactive group such as an amino, hydroxyl or sulfhydryl group. The present invention may be used in single- and two-step labeling processes. In the two-step labeling process, a primary component, such as an oligonucleotide is labeled with the reagent capable of introducing the novel fluorophore PPT-, coumarin- and resorufin-based dyes, by reaction with a reactive group of the ODN (such as an amine, hydroxyl, carboxyl, aldehyde or sulfhydryl group) and the label is used to probe for a secondary component, such as an oligonucleotide target.